Compound for preparing myocardial cells and application thereof

Abstract

Relates to the field of biomedicine, in particular to the field of regenerative medicine. Specifically, the invention relates to a compound for preparing myocardial cells and application thereof, and a method for differentiating pluripotent stem cells such as induced pluripotent stem cells into myocardial cells by using the compound.

Description

Compounds for preparing myocardial cells and uses thereof Technical Field

[0001] The present invention relates to the field of biomedicine, in particular to the field of regenerative medicine. Specifically, the present invention relates to a compound for preparing cardiomyocytes and its use, as well as a method for differentiating pluripotent stem cells, such as induced pluripotent stem cells, into cardiomyocytes using the compound.

[0002] Background of the Invention

[0003] Heart disease is one of the leading causes of death in humans, and cardiac cell therapy, in vitro myocardial disease modeling, or drug development all require the support of a large number of cardiomyocytes. However, cardiomyocytes, as terminally differentiated cells, cannot regenerate in vivo, and primary cardiomyocytes are difficult to culture and expand in vitro. Therefore, the source of cardiomyocytes has become a major problem. At present, induced pluripotent stem cells (hiPSCs) can be obtained by reprogramming human somatic cells, thereby directing differentiation into a variety of functional cells, including cardiomyocytes (hiPSC-CMs), which provides a new idea for the source of cardiomyocytes. The efficient, stable, and large-scale production of hiPSC-CMs is crucial for their practical application, but the efficiency and quality of the current differentiation system are still unstable. Therefore, there is still a need in the art for new compounds and methods that can be used to efficiently generate functional cardiomyocytes from stem cells, such as induced pluripotent stem cells, by reprogramming, thereby treating heart diseases such as heart failure.

[0004] SUMMARY OF THE INVENTION

[0005] The present invention provides at least the following embodiments:

[0006] Embodiment 1. A method for preparing cardiomyocytes by differentiating pluripotent stem cells, the method comprising:

[0007] i) providing pluripotent stem cells;

[0008] ii) culturing the pluripotent stem cells in a basal medium supplemented with a CDK8 inhibitor and a WNT signaling pathway activator for about 24 hours to about 48 hours, and then continuing to culture the cells in a basal medium without the CDK8 inhibitor and the WNT signaling pathway activator for about 24 hours;

[0009] iii) culturing the cells obtained in step ii) in a basal medium supplemented with a WNT signaling pathway inhibitor for approximately 48 hours, and then continuing to culture the cells in a basal medium without the WNT signaling pathway inhibitor for approximately 24 hours;

[0010] iv) culturing the cells obtained in step iii) in a basal medium supplemented with insulin for about 3-6 days; and

[0011] v) Optionally, harvesting the obtained cardiomyocytes.

[0012] Embodiment 2. The method of embodiment 1, wherein the pluripotent stem cell is selected from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0013] Embodiment 3. The method of embodiment 1 or 2, wherein the pluripotent stem cells in step i) are cultured in a multi-well plate.

[0014] Embodiment 4. The method of any one of embodiments 1-3, wherein the pluripotent stem cells in step i) are grown to a confluence of about 80-90%.

[0015] Embodiment 5. The method of any one of embodiments 1-4, wherein the basal culture medium is a combination of RPMI medium and insulin-free B27 cell culture supplement.

[0016] Embodiment 6. The method according to any one of embodiments 1 to 6, wherein the basal medium supplemented with insulin in step iv) is a combination of RPMI medium (such as RPMI1640 medium) and B27 cell culture supplement.

[0017] Embodiment 7. The method of any one of embodiments 1-6, wherein the CDK8 inhibitor is selected from BI-1347 or a structural analog thereof, MSC2530818 or a structural analog thereof, AS2863619 or a structural analog thereof, Senexin A or a structural analog thereof, CCT-251921 or a structural analog thereof, Senexin A or a structural analog thereof, C or a structural analog thereof, JH-XVI-178 or a structural analog thereof, CCT251545 or a structural analog thereof, BRD6989 or a structural analog thereof, JH-XI-10-02 or a structural analog thereof, SEL120-34A or a structural analog thereof, LY2857785 or a structural analog thereof, CDK8-IN-1 or a structural analog thereof, CDK8-IN-5 or a structural analog thereof, CDK8-IN-3 or a structural analog thereof, CDK8-IN-4 or a structural analog thereof, CDK8-IN-6 or a structural analog thereof, CDK8-IN-7 or a structural analog thereof, CDK8 / 19-IN-1 or a structural analog thereof, DS96432529 or a structural analog thereof, Wogonin or a structural analog thereof, preferably, the CDK8 inhibitor is BI-1347 or a structural analog thereof.

[0018] Embodiment 8. The method of any one of Embodiments 1-7, wherein the WNT signaling pathway activator is CHIR99021 or a structural analog thereof, CHIR98014 or a structural analog thereof, or SB212763 or a structural analog thereof.

[0019] Embodiment 9. The method of any one of embodiments 1-8, wherein the WNT signaling pathway inhibitor is IWR1 or a structural analog thereof, IWP2 or a structural analog thereof, or IWP4 or a structural analog thereof.

[0020] Embodiment 10. The method of any one of embodiments 1-9, wherein the insulin is human insulin.

[0021] Embodiment 11. The method of any one of embodiments 1-10, wherein the final concentration of the CDK8 inhibitor, such as BI-1347 or a structural analog thereof, is about 0.001 μM to about 5 μM, preferably about 0.004 μM to about 3 μM, such as about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM or about 5 μM.

[0022] Embodiment 12. The method of any one of Embodiments 1-11, wherein the final concentration of the WNT signaling pathway activator, such as CHIR99021 or a structural analog thereof, is about 1 μM to about 25 μM or higher, for example, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, greater than 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM or higher.

[0023] Embodiment 13. The method of any one of Embodiments 1-12, wherein the concentration of the WNT signaling pathway inhibitor is about 1 μM to about 10 μM or higher, for example, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM or higher.

[0024] Embodiment 14. The method of any one of embodiments 1-13, wherein the pluripotent stem cells are human pluripotent stem cells and the cardiomyocytes are human cardiomyocytes.

[0025] Embodiment 15. The method of any one of embodiments 1-14, wherein the cardiomyocytes are functional cardiomyocytes, eg, cardiomyocytes capable of beating.

[0026] Embodiment 16. The method of any one of embodiments 1-15, wherein the cardiomyocytes express one or more cardiac-specific proteins selected from the group consisting of cTNT, cTNI, α-ACTININ, MEF2C, and NKX2.5.

[0027] Embodiment 17. A cardiomyocyte prepared by the method of any one of embodiments 1-16.

[0028] Embodiment 18. A pharmaceutical composition comprising cardiomyocytes prepared by the method of any one of embodiments 1-16 and a pharmaceutically acceptable carrier.

[0029] Embodiment 19. Use of the cardiomyocytes of embodiment 17 or the pharmaceutical composition of embodiment 18 in the preparation of a medicament for treating a cardiac disease in a subject in need thereof.

[0030] Embodiment 20. A method of treating cardiac disease in a subject in need thereof, comprising administering to the subject the cardiomyocytes of embodiment 17 or the pharmaceutical composition of embodiment 18.

[0031] Embodiment 21. The use of embodiment 19 or the method of embodiment 20, wherein the subject is a human.

[0032] Embodiment 22. Use of a CDK8 inhibitor in the preparation of a reagent for preparing cardiomyocytes by differentiation of pluripotent stem cells.

[0033] Embodiment 23. The use of embodiment 22, wherein the CDK8 inhibitor is selected from BI-1347 or a structural analog thereof, MSC2530818 or a structural analog thereof, AS2863619 or a structural analog thereof, Senexin A or a structural analog thereof, CCT-251921 or a structural analog thereof, Senexin C or a structural analog thereof, JH-XVI-178 or a structural analog thereof, CCT251545 or a structural analog thereof, BRD6989 or a structural analog thereof, JH-XI-10-02 or a structural analog thereof, SEL120-34A or a structural analog thereof, LY2857785 or a structural analog thereof, CDK8-IN-1 or a structural analog thereof, CDK8-IN-5 or a structural analog thereof, CDK8-IN-3 or a structural analog thereof, CDK8-IN-4 or a structural analog thereof, CDK8-IN-6 or a structural analog thereof, CDK8-IN-7 or a structural analog thereof, CDK8 / 19-IN-1 or a structural analog thereof, DS96432529 or a structural analog thereof, Wogonin or a structural analog thereof, preferably, the CDK8 inhibitor is BI-1347 or a structural analog thereof.

[0034] Embodiment 24. The use according to embodiment 22 or 23, wherein the agent is used in combination with a WNT signaling pathway activator, a WNT signaling pathway inhibitor and / or insulin to prepare cardiomyocytes by differentiating pluripotent stem cells.

[0035] Embodiment 25. The use according to any one of embodiments 22 to 24, wherein the agent is used to improve the efficiency of preparing cardiomyocytes by differentiation from pluripotent stem cells.

[0036] Embodiment 26. The use according to any one of embodiments 22-25, wherein the agent is used to improve the efficiency of preparing cardiomyocytes by differentiating pluripotent stem cells in the presence of a high concentration of a WNT signaling pathway activator.

[0037] Embodiment 27. The use according to any one of embodiments 22-26, wherein the reagent is used to prepare cardiomyocytes by the method according to any one of embodiments 1-16.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1. The human stem cell to cardiomyocyte differentiation process used in this experiment. The differentiation process is divided into four stages: hiPSC stage, stage 1 differentiation to mesoderm, stage 2 differentiation to cardiac progenitors, and stage 3 differentiation to cardiomyocytes. This process is primarily accomplished using WNT signaling pathway activators (CHIR) and inhibitors (IWR1). The colored arrows indicate that the duration and concentration of the small molecule CHIR application during stage 1 significantly influence differentiation efficiency.

[0040] Figure 2. High-dose CHIR group, adding BI-1347 between 0 and 48 hours resulted in highly efficient cardiomyocyte differentiation. (a) Difference in differentiation efficiency between the high-dose CHIR group (CHIR 20 μM between 0 and 48 hours) with and without BI-1347 in the first stage. Green represents cardiomyocytes positive for cTNT immunofluorescence staining, and blue represents the nuclear dye Hoechst. (b) Statistical results of cardiomyocyte differentiation efficiency compared with BI-1347 and two other compounds with the same target (MSC2530818 and AS2863619) at different CHIR concentrations. The results were similar among the three compounds, with the high-dose group achieving significantly higher differentiation efficiency than the control group. (c) Scale bar represents 1 mm.

[0041] Figure 3. Validation of BI-1347's effect on a stable and optimized cardiomyocyte system. (a) Effect of different CHIR concentrations in the first phase on the differentiation efficiency of the hiPSC-M cell line with or without BI-1347 (0-48h, 0.5μM). (b) Effect of different CHIR concentrations in the first phase on the differentiation efficiency of the hiPSC-F cell line with or without BI-1347 (0-48h, 0.5μM). (c) Differentiation efficiency at different BI-1347 concentrations. (d) Effect of different BI-1347 doses on differentiation efficiency. In the first phase, cells were treated with a high CHIR concentration and Compound A for 0-48h.

[0042] Figure 4. Characterization of hiPSC-CM gene transcriptional expression after the BI-1347-optimized system. (a) Principal component analysis of bulk RNA-seq data for iPSCs, iPSC-cm-BI-1347, and iPSC-cm + BI-1347 (0.5 μM, 48 hours). Each dot represents a single RNA-seq sample. N = 3. (b) Genome-wide comparison of iPSCs, iPSC-cm, and iPSC-cm treated with BI-1347 (0-48 hours) without metabolic purification. The heat map shows log2(FPKM+1)-normalized z-scores across multiple samples. Hierarchical clustering was performed for 19,999 genes. (c) Gene expression data for iPSCs, iPSC-cm without BI-1347 treatment, and iPSC-cm treated with BI-1347 (0.5 μM, 48 hours) (day 12, all without metabolic purification). The heatmap displays log2(FPKM+1)-normalized z-scores across multiple samples. (d) Immunofluorescence analysis of hiPSC-CMs on day 12 of differentiation following addition of BI-1347 in stage 1. From left to right: cTNT (red), MEF2C (green), and the nuclear dye Hoechst (blue); NKX2.5 (red), cTNI (green), and the nuclear dye Hoechst (blue); and α-ACTININ (green), and the nuclear dye Hoechst (blue). Scale bars for all three images are 100 μm.

[0043] Figure 5. qPCR characterization of hiPSC-CMs using the optimized BI-1347 system. (a) Action potentials in iPSC-CMs cultured with BI-1347 (0.5 μmol / L, 0-48 h) and without BI-1347. (b) Current-voltage plots of calcium currents in iPSC-CMs cultured with and without BI-1347. Data are presented as mean ± SEM. n = 15-16. (c) Comparison of the maximum diastolic voltage (MDP), peak voltage, action potential amplitude (APA), and time to repolarization to different levels (90% and 50%) of the action potential between the BI-1347-treated group (n = 25 cells) and the BI-1347-untreated group (n = 25 cells). (d) Recording procedures and examples of calcium currents recorded in voltage-clamp mode between the BI-1347-treated group and the BI-1347-untreated group. (e) Calcium current density recorded from the two groups of iPSC-CMs at a clamping voltage of -10 mV. (f) Action potential graphs of the three iPSC-CM subtypes recorded from the BI-1347-treated group (n = 25 cells) and the BI-1347-untreated group (n = 25 cells). (g) Percentage of iPSC-CM subtypes. (h) Comparison of the maximum diastolic voltage (MDP), peak voltage, action potential amplitude (APA), and time for action potential repolarization to different levels (90% and 50%) in the ventricular myoid cells cultured with BI-1347 (n=19 cells), ventricular myoid cells cultured without BI-1347 (n=21 cells), atrial myoid cells cultured with BI-1347 (n=3 cells), atrial myoid cells cultured without BI-1347 (n=2 cells), sinoatrial node-like cells cultured with BI-1347 (n=3 cells), and sinoatrial node-like cells cultured without BI-1347 (n=2 cells). (i) Comparison of spontaneous calcium transient fluorescence intensity, time to maximum, full width at half maximum, frequency, time to 50% of maximum, and time for calcium transient decay to 50% and 90%, respectively, between the BI-1347-treated group (n = 39 cells) and the BI-1347-untreated group (n = 40 cells). (j) Fluorescence images (top) and time course (bottom) of spontaneous calcium transients in iPSC-CMs cultured with and without BI-1347. DETAILED DESCRIPTION

[0044] In the present invention, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and routine procedures widely used in the corresponding fields. For example, standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following literature: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook").

[0045] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0046] In one aspect, the present invention provides a CDK8 inhibitor for use in differentiating pluripotent stem cells to produce cardiomyocytes. In some embodiments, the CDK8 inhibitor is used in differentiating pluripotent stem cells to produce cardiomyocytes in vitro.

[0047] In one aspect, the present invention provides a use of a CDK8 inhibitor as described herein in the preparation of a reagent for preparing cardiomyocytes by differentiating pluripotent stem cells. In some embodiments, the reagent is used for preparing cardiomyocytes by differentiating pluripotent stem cells in vitro.

[0048] The pluripotent stem cells described herein can be pluripotent stem cells from mammals, for example, mice, rats, non-human primates or humans. Preferably, the pluripotent stem cells described herein are human pluripotent stem cells. The pluripotent stem cells described herein include but are not limited to embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). Preferably, the pluripotent stem cells described herein are induced pluripotent stem cells (iPSC). Preferably, the pluripotent stem cells described herein are isolated pluripotent stem cells.

[0049] The embryonic stem cells (ESCs) described herein can be derived from humans or non-human animals. Human embryonic stem cells (ESCs) are limited to stem cells isolated from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0050] iPSC described herein can be derived from any cell type. For example, the starting cell type can be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, hepatocytes or gastric cells. The induction of iPSC has no restrictions on the degree of cell differentiation or the age of the object from which the cells are collected; even undifferentiated progenitor cells (including adult stem cells) and terminally differentiated mature cells can be used as the source of iPSC herein. Those skilled in the art can easily apply methods known in the art to produce induced pluripotent stem cells. For example, reprogramming factors can be used to produce pluripotent stem cells from somatic cells. However, it is also possible to use small molecule compounds to induce pluripotent stem cells from somatic cells. Alternatively, pluripotent stem cells can be induced from somatic cells by a combination of reprogramming factors and small molecule compounds.

[0051] In some embodiments of the aspects herein, the cardiomyocytes are human cardiomyocytes. In some embodiments of the aspects herein, the cardiomyocytes are functional cardiomyocytes. In some embodiments of the aspects herein, the cardiomyocytes are beating cardiomyocytes. In some embodiments of the aspects herein, the cardiomyocytes express one or more cardiac-specific proteins selected from the group consisting of cTNT, cTNI, α-ACTININ, MEF2C, and NKX2.5.

[0052] In some embodiments of the aspects herein, the CDK8 inhibitor or the agent is used in combination with a WNT signaling pathway activator, a WNT signaling pathway inhibitor, and / or insulin to prepare cardiomyocytes by differentiating pluripotent stem cells.

[0053] In some embodiments of various aspects herein, the CDK8 inhibitor or the reagent is used to improve the efficiency of preparing cardiomyocytes by differentiation of pluripotent stem cells. In some embodiments, the CDK8 inhibitor or the reagent is used to improve the efficiency of preparing cardiomyocytes by differentiation of pluripotent stem cells in the presence of high concentrations of WNT signaling pathway activators (e.g., about 8 μM-about 25 μM or higher concentrations of WNT signaling pathway activators such as CHIR99021).

[0054] In some embodiments, the concentration of the WNT signaling pathway activator is about 1 μM to about 25 μM or more, for example, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, greater than 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM or more.

[0055] In some embodiments of the various aspects herein, the CDK8 inhibitor is used at a concentration of about 0.001 μM to about 5 μM, preferably about 0.004 μM to about 3 μM, such as about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, or about 5 μM.

[0056] In some embodiments of the aspects herein, the concentration of the WNT signaling pathway inhibitor is about 1 μM to about 10 μM or higher, such as about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM or higher.

[0057] In some embodiments of the various aspects herein, the use of the CDK8 inhibitor can increase the efficiency of preparing cardiomyocytes by differentiating pluripotent stem cells (particularly when using a WNT signaling pathway activator such as CHIR99021 at a concentration of about 8 μM to about 25 μM or more) by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500% or more compared to not using the CDK8 inhibitor.

[0058] In some embodiments of the various aspects herein, the CDK8 inhibitors include but are not limited to BI-1347 or a structural analog thereof, MSC2530818 or a structural analog thereof, AS2863619 or a structural analog thereof, Senexin A or a structural analog thereof, CCT-251921 or a structural analog thereof, Senexin A or a structural analog thereof, C or a structural analog thereof, JH-XVI-178 or a structural analog thereof, CCT251545 or a structural analog thereof, BRD6989 or a structural analog thereof, JH-XI-10-02 or a structural analog thereof, SEL120-34A or a structural analog thereof, LY2857785 or a structural analog thereof, CDK8-IN-1 or a structural analog thereof, CDK8-IN-5 or a structural analog thereof, CDK8-IN-3 or a structural analog thereof, CDK8-IN-4 or a structural analog thereof, CDK8-IN-6 or a structural analog thereof, CDK8-IN-7 or a structural analog thereof, CDK8 / 19-IN-1 or a structural analog thereof, DS96432529 or a structural analog thereof, Wogonin or a structural analog thereof. Preferably, the CDK8 inhibitor is BI-1347 or a structural analog thereof.

[0059] More CDK8 inhibitors suitable for the present invention can be found in the literature, for example: Yu, Mingfeng, et al. "Potent and orally bioavailable CDK8 inhibitors: Design, synthesis, structure-activity relationship analysis and biological evaluation." European Journal of Medicinal Chemistry 214(2021): 113248; Bhurta, Deendyal, and Sandip B. Bharate. "Analyzing the scaffold diversity of cyclin-dependent kinase inhibitors and revisiting the clinical and preclinical pipeline." Medicinal Research Reviews 42.2(2022): 654-709; and Hatcher, John M., et al. "Development of Highly Potent and Selective Pyrazolopyridine Inhibitor of CDK8 / 19." ACS Medicinal Chemistry Letters 12.11(2021): 1689-1693.

[0060] In some embodiments of various aspects herein, the WNT signaling pathway activator includes but is not limited to CHIR99021 or a structural analog thereof, CHIR98014 or a structural analog thereof, SB212763 or a structural analog thereof. In some embodiments, the WNT signaling pathway inhibitor includes but is not limited to IWR1 or a structural analog thereof, IWP2 or a structural analog thereof, IWP4 or a structural analog thereof. In some embodiments, the insulin is human insulin.

[0061] The use of CDK8 inhibitors, WNT signaling pathway activators and / or WNT signaling pathway inhibitors in the present invention encompasses their salt forms.

[0062] In some embodiments of the aspects herein, the CDK8 inhibitor is used to prepare cardiomyocytes by differentiating pluripotent stem cells by a method comprising the following steps:

[0063] i) providing pluripotent stem cells;

[0064] ii) culturing the pluripotent stem cells in a basal medium supplemented with a CDK8 inhibitor and a WNT signaling pathway activator;

[0065] iii) culturing the cells obtained in step ii) in a basal medium supplemented with a WNT signaling pathway inhibitor;

[0066] iv) culturing the cells obtained in step iii) in a basal medium supplemented with insulin; and

[0067] v) Optionally, harvesting the obtained cardiomyocytes.

[0068] In some embodiments, step ii) includes culturing the pluripotent stem cells for a first period of time in a basal medium supplemented with a CDK8 inhibitor and a WNT signaling pathway activator, and then continuing to culture the cells for a second period of time in a basal medium not supplemented with the CDK8 inhibitor and the WNT signaling pathway activator. In some embodiments, the first period of time is about 24 hours (h) to about 48 hours (h). In some embodiments, the second period of time is about 24 hours (h).

[0069] In some embodiments, step iii) includes culturing the cells obtained in step ii) in a basal medium supplemented with a WNT signaling pathway inhibitor for a third time period, and then continuing to culture the cells in a basal medium without the addition of a WNT signaling pathway inhibitor for a fourth time period. In some embodiments, the third time period is about 24 hours (h) to about 48 hours (h). In some embodiments, the fourth time period is about 24 hours (h).

[0070] In some embodiments, step iv) comprises culturing the cells obtained in step iii) in a basal medium supplemented with insulin for a fifth period of time. In some embodiments, the fifth period of time is about 3 days to about 6 days.

[0071] In some embodiments, the method comprises:

[0072] i) providing pluripotent stem cells;

[0073] ii) culturing the pluripotent stem cells in a basal medium supplemented with the CDK8 inhibitor and the WNT signaling pathway activator for about 24 hours to about 48 hours, and then continuing to culture the cells in a basal medium without the CDK8 inhibitor and the WNT signaling pathway activator for about 24 hours;

[0074] iii) culturing the cells obtained in step ii) in a basal medium supplemented with the WNT signaling pathway inhibitor for approximately 48 hours, and then continuing to culture the cells in a basal medium without the WNT signaling pathway inhibitor for approximately 24 hours;

[0075] iv) culturing the cells obtained in step iii) in a basal medium supplemented with insulin for about 3-6 days; and

[0076] v) Optionally, harvesting the obtained cardiomyocytes.

[0077] In some embodiments, the pluripotent stem cells in step i) are cultured in a multi-well plate. In some embodiments, the pluripotent stem cells in step i) are grown to a confluence of about 80-90%.

[0078] In some embodiments, the basal medium is a combination of RPMI medium (such as RPMI1640 medium) and B27 cell culture supplement (B27minus insulin) without insulin; or a combination of CDM3 medium (Paul W Burridge et al., Chemically defined generation of human cardiomyocytes. Nature Methods volume 11, pages855–860 (2014)) and S12 cell culture supplement (Fei Pei et al., Chemical-defined and albumin-free generation of human atrial and ventricular myocytes from human pluripotent stem cells. Stem Cell Research, Volume 19, March 2017, Pages 94-103). In some embodiments, the basal medium to which insulin is added in step iv) is a combination of RPMI medium (such as RPMI1640 medium) and B27 cell culture supplement, or a combination of CDM3 medium and S12 cell culture supplement. In some embodiments, the volume ratio of RPMI medium and B27 cell culture supplement (with or without insulin) in the base medium is about 50:1.

[0079] In some embodiments, the culture medium in step iii) does not contain the CDK8 inhibitor and the WNT signaling pathway activator. In some embodiments, the culture medium in step iv) does not contain the CDK8 inhibitor, the WNT signaling pathway activator and the WNT signaling pathway inhibitor.

[0080] In some embodiments, the cells obtained in step ii) are primarily cardiac mesoderm cells. In some embodiments, the cells obtained in step iii) are primarily cardiac progenitor cells. In some embodiments, the cells obtained in step iv) are primarily cardiomyocytes.

[0081] In another aspect, the present invention provides a method for preparing cardiomyocytes by differentiating pluripotent stem cells, the method comprising:

[0082] i) providing pluripotent stem cells;

[0083] ii) culturing the pluripotent stem cells in a basal medium supplemented with a CDK8 inhibitor and a WNT signaling pathway activator;

[0084] iii) culturing the cells obtained in step ii) in a basal medium supplemented with a WNT signaling pathway inhibitor;

[0085] iv) culturing the cells obtained in step iii) in a basal medium supplemented with insulin; and

[0086] v) Optionally, harvesting the obtained cardiomyocytes.

[0087] In some embodiments, step ii) includes culturing the pluripotent stem cells for a first period of time in a basal medium supplemented with a CDK8 inhibitor and a WNT signaling pathway activator, and then continuing to culture the cells for a second period of time in a basal medium not supplemented with the CDK8 inhibitor and the WNT signaling pathway activator. In some embodiments, the first period of time is about 24 hours (h) to about 48 hours (h). In some embodiments, the second period of time is about 24 hours (h).

[0088] In some embodiments, step iii) includes culturing the cells obtained in step ii) in a basal medium supplemented with a WNT signaling pathway inhibitor for a third time period, and then continuing to culture the cells in a basal medium without the addition of a WNT signaling pathway inhibitor for a fourth time period. In some embodiments, the third time period is about 24 hours (h) to about 48 hours (h). In some embodiments, the fourth time period is about 24 hours (h).

[0089] In some embodiments, step iv) comprises culturing the cells obtained in step iii) in a basal medium supplemented with insulin for a fifth period of time. In some embodiments, the fifth period of time is about 3 days to about 6 days.

[0090] In some embodiments, the method comprises:

[0091] i) providing pluripotent stem cells;

[0092] ii) culturing the pluripotent stem cells in a basal medium supplemented with a CDK8 inhibitor and a WNT signaling pathway activator for about 24 hours (h) to about 48 hours (h), and then continuing to culture the cells in a basal medium without the CDK8 inhibitor and the WNT signaling pathway activator for about 24 hours;

[0093] iii) culturing the cells obtained in step ii) in a basal medium supplemented with a WNT signaling pathway inhibitor for approximately 48 hours, and then continuing to culture the cells in a basal medium without the WNT signaling pathway inhibitor for approximately 24 hours;

[0094] iv) culturing the cells obtained in step iii) in a basal medium supplemented with insulin for about 3-6 days; and

[0095] v) Optionally, harvesting the obtained cardiomyocytes.

[0096] In some embodiments, the pluripotent stem cells in step i) are cultured in a multi-well plate. In some embodiments, the pluripotent stem cells in step i) are grown to a confluence of about 80-90%.

[0097] In some embodiments, the basal medium is a combination of RPMI medium (such as RPMI1640 medium) and a B27 cell culture supplement that does not contain insulin; or a combination of CDM3 medium and an S12 cell culture supplement that does not contain insulin. In some embodiments, the basal medium supplemented with insulin in step iv) is a combination of RPMI medium (such as RPMI1640 medium) and a B27 cell culture supplement, or a combination of CDM3 medium and an S12 cell culture supplement.

[0098] In some embodiments, the cells obtained in step ii) are primarily cardiac mesoderm cells. In some embodiments, the cells obtained in step iii) are primarily cardiac progenitor cells. In some embodiments, the cells obtained in step iv) are primarily cardiomyocytes.

[0099] In some embodiments, the CDK8 inhibitors include but are not limited to BI-1347 or a structural analog thereof, MSC2530818 or a structural analog thereof, AS2863619 or a structural analog thereof, Senexin A or a structural analog thereof, CCT-251921 or a structural analog thereof, Senexin A or a structural analog thereof, C or a structural analog thereof, JH-XVI-178 or a structural analog thereof, CCT251545 or a structural analog thereof, BRD6989 or a structural analog thereof, JH-XI-10-02 or a structural analog thereof, SEL120-34A or a structural analog thereof, LY2857785 or a structural analog thereof, CDK8-IN-1 or a structural analog thereof, CDK8-IN-5 or a structural analog thereof, CDK8-IN-3 or a structural analog thereof, CDK8-IN-4 or a structural analog thereof, CDK8-IN-6 or a structural analog thereof, CDK8-IN-7 or a structural analog thereof, CDK8 / 19-IN-1 or a structural analog thereof, DS96432529 or a structural analog thereof, Wogonin or a structural analog thereof. Preferably, the CDK8 inhibitor is BI-1347 or a structural analog thereof. In some embodiments, the WNT signaling pathway activator includes but is not limited to CHIR99021 or a structural analog thereof, CHIR98014 or a structural analog thereof, SB212763 or a structural analog thereof. In some embodiments, the WNT signaling pathway inhibitor includes but is not limited to IWR1 or a structural analog thereof, IWP2 or a structural analog thereof, IWP4 or a structural analog thereof. In some embodiments, the insulin is human insulin.

[0100] In some embodiments, the final concentration of the CDK8 inhibitor, such as BI-1347 or a structural analog thereof, is about 0.001 μM to about 5 μM, preferably about 0.004 μM to about 3 μM, such as about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM or about 5 μM.

[0101] In some embodiments, the final concentration of the WNT signaling pathway activator, such as CHIR99021 or a structural analog thereof, is about 1 μM to about 25 μM or higher, for example, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, greater than 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM or higher.

[0102] In some embodiments, the concentration of the WNT signaling pathway inhibitor is about 1 μM to about 10 μM or higher, such as about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM or higher.

[0103] In one aspect, the present invention provides a cardiomyocyte prepared by the method of the present invention.

[0104] In one aspect, the present invention provides a pharmaceutical composition comprising cardiomyocytes prepared by the method of the present invention and a pharmaceutically acceptable carrier.

[0105] In one aspect, the present invention further provides use of the cardiomyocytes prepared by the method of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a heart disease in a subject in need thereof. The heart disease, in particular myocardial disease, includes but is not limited to heart failure, myocardial infarction, and the like.

[0106] In one aspect, the present invention also provides a method for treating heart disease in a subject in need thereof, comprising administering to the subject the cardiomyocytes prepared by the method of the present invention or the pharmaceutical composition of the present invention.

[0107] As used herein, a "subject" can be a mammal or a non-mammal. The subject can be a human, or a non-human mammal such as a mouse or rat or a non-human primate, preferably a human.

[0108] On the other hand, the present invention provides a culture medium for preparing cardiomyocytes by differentiating pluripotent stem cells, the culture medium comprising the CDK8 inhibitor and the WNT signaling pathway activator. In some embodiments, the culture medium further comprises a basal medium, for example, the basal medium is a combination of RPMI culture medium (such as RPMI1640 culture medium) and a B27 cell culture additive without insulin; or a combination of CDM3 culture medium and an S12 cell culture additive without insulin.

[0109] In another aspect, the present invention provides a culture medium system for preparing cardiomyocytes by differentiating pluripotent stem cells, the culture medium system comprising

[0110] 1) a first culture medium comprising the CDK8 inhibitor and the WNT signaling pathway activator described herein;

[0111] 2) a second culture medium comprising a WNT signaling pathway inhibitor as described herein; and

[0112] 3) A third culture medium comprising insulin.

[0113] In some embodiments, the first, second and / or third culture medium further comprises a basal culture medium, for example, the basal culture medium is a combination of RPMI medium (such as RPMI1640 medium) and B27 cell culture supplement without insulin; or a combination of CDM3 medium and S12 cell culture supplement without insulin.

[0114] In some embodiments, the culture medium system further comprises a fourth culture medium for culturing and / or expanding pluripotent stem cells. The fourth culture medium can be any culture medium that can be used for culturing and / or expanding pluripotent stem cells and is readily available to those skilled in the art.

[0115] In some embodiments, the culture medium or culture medium system is used in the methods of the present invention.

[0116] In one aspect, the present invention provides a kit for preparing cardiomyocytes by differentiating pluripotent stem cells, the kit comprising at least a CDK8 inhibitor as defined herein. In some embodiments, the kit comprises a culture medium or culture medium system of the present invention.

[0117] Example

[0118] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the relevant specific embodiments and accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0119] Example 1. Differentiation of human stem cells into cardiomyocytes

[0120] The main process of human stem cell differentiation into cardiomyocytes is shown in Figure 1 (see Aguilar et al., 2015). It involves culturing human hiPSC cells in a monolayer and starting differentiation when the confluence reaches about 80%. In the first stage (0-72h), the WNT signaling pathway activator CHIR99021 (CHIR) is used; in the second stage, the WNT signaling pathway inhibitor IWR1 is treated for 48h; in the third stage, insulin is added to the basal differentiation medium, and the cells can spontaneously differentiate into beating cardiomyocytes. The entire process goes through four stages: stem cells (hiPSC), cardiac mesoderm (Cardiac mesoderm, Stage I), cardiac progenitor cells (CPC, Stage II), and cardiomyocytes (CM, Stage III). Under normal circumstances, beating cardiomyocytes can be observed under a microscope within 7-10 days. More detailed experimental steps are as follows:

[0121] The main reagents used in the experiment are shown in the table below:

[0122] 1.1 Passaging and culture of stem cells

[0123] The hiPSCs used in this experiment were routinely cultured in 6-well plates and passaged approximately every four days in a cell culture incubator maintained at 37°C and 5% CO2. The passage steps are detailed below:

[0124] 1) Observe the cell density under a microscope. When the cells have proliferated to about 80% of the total area, they are ready for subculture.

[0125] 2) Before passaging, pre-spread Matrigel in the well plate. All Matrigel operations must be performed on ice. Dilute the original Matrigel 50-fold with pre-chilled DMEM / F-12 and add enough to cover the bottom of the well plate (850 μL / well for a 6-well plate, for example). Incubate in a 37°C incubator for 30 minutes. Aspirate the liquid before use.

[0126] 3) Preheat the stem cell culture medium PGM1 or CDM (depending on the purpose of the subsequent experiment), DPBS, and Versene to 37°C in advance, and add Y27632 (5 μM) to the stem cell culture medium;

[0127] 4) After removing the cells from the incubator, aspirate the culture medium. Add 1 ml of DPBS to each well to wash the remaining culture medium, aspirate it, add 1 ml of Versene, and digest at 37°C for 3 minutes.

[0128] 5) Immediately remove the Versene and pipette the cells 3-4 times with 1 ml of PGM1 medium to release the cells from the bottom of the dish.

[0129] 6) Mix the cell suspension with the remaining culture medium and add to a new plate covered with Matrigel. The cell culture ratio should be 1:6-1:12, but this ratio may be adjusted slightly depending on the starting cell number.

[0130] 7) Replace the PGM1 medium with fresh one 12-24 hours after passage to remove the Y27632 medium. Observe the cell status and density daily.

[0131] 1.2 Cardiomyocyte differentiation

[0132] Stem cell to cardiomyocyte differentiation is routinely performed in 24-, 96-, or 384-well plates. The steps are detailed below (Figure 3.1):

[0133] 1) hiPSCs were separated into CDM medium at a ratio of 1:10 or 1:12. The separation steps were the same as the above passaging steps. Y27632 (5 μM) was added to the CDM medium. The day was recorded as -3.

[0134] 2) 12-24 hours after passage, change the medium to remove Y27632 and continue culturing in CDM medium. Observe the cell status and density daily.

[0135] 3) When hiPSCs reached 80-90% confluence, the culture medium was replaced with RPMI+B27minus (50 ml RPMI1640 + 1 ml B27minus) and various concentrations of CHIR were added, with or without the addition of various concentrations of BI-1347. This was designated as differentiation day 0, marking the end of the hiPSC stage.

[0136] Note: Without BI-1347, the dosage of CHIR is flexible and unstable. Different cell lines, different batches, and different operators can lead to large differences in differentiation results.

[0137] 4) After 24-48 hours, the culture medium was replaced with RPMI+B27minus;

[0138] 5) At 72 h, the culture medium was replaced with RPMI+B27minus and 5 μM of the small molecule IWR1 was added. This was recorded as the third day of differentiation. At this time, the cells differentiated into the mesoderm stage, and the first stage ended.

[0139] 6) 48 hours after IWR1 addition, the culture medium was replaced with RPMI+B27minus and IWR1 was removed, marking the 5th day of differentiation;

[0140] 7) Culture the cells in RPMI+B27minus for 24 hours, which is recorded as differentiation day 6. At this time, the cells differentiate into hiPSC-CPCs. Subsequently, the culture medium is replaced with RPMI+B27, and the second stage is completed.

[0141] 8) Continue culturing in RPMI + B27 medium, changing the medium every 3 days. The cells will spontaneously differentiate into beating hiPSC-CMs within 3-6 days, marking the third stage of differentiation. Cell beating can be observed as early as day 7-8.

[0142] Example 2: Improving hiPSC cardiomyogenic differentiation efficiency using the small molecule BI-1347 at high CHIR doses

[0143] The inventors surprisingly found that in the process of differentiating human stem cells into cardiomyocytes, the dose of CHIR99021 (CHIR) in the first stage (from hiPSC to cardiac mesoderm, 0-72h) plays a decisive role in whether the differentiation is successful. Under the premise of appropriate starting cell density, the optimal concentration of CHIR, a WNT pathway activator used in the first stage of differentiation, is unstable, and there are significant differences between cell lines or batches. Moreover, the optimal concentration range of CHIR is very narrow, and often only a concentration range of 2-4 μM can achieve a higher differentiation efficiency. The above problems make the differentiation system very unstable and also make the large-scale production of cardiomyocytes challenging.

[0144] The inventors surprisingly discovered that the addition of the small molecule BI-1347 during the first phase (0-48 hours) effectively improved the cardiomyocyte differentiation efficiency of various hiPSC cell lines across different CHIR dose groups, particularly the high CHIR dose group (Figures 2a, 2b, 3a, 3b). This significantly broadened the effective concentration of CHIR, and beating cardiomyocytes were visible after approximately 7-10 days of differentiation. After the addition of BI-1347, the standard deviation of cardiomyocyte differentiation efficiency was significantly reduced and the mean significantly improved across batches of the same cell line or across different cell lines, with very few batches experiencing complete differentiation failure (Figures 2c, 2d).

[0145] BI-1347 has a stable and outstanding effect in multiple batches of experiments on the same cell line. The results of the effective concentration test showed that BI-1347 has a significant effect in a wide concentration range (0.004-3μM) (Figure 3c, Figure 3d). In addition, since the target of BI-1347 (CDK8 inhibitor) is known, two other compounds with the same target (MSC2530818 and AS2863619) were also tested. Their effects on myocardial differentiation efficiency are similar to those of BI-1347 (Figure 2b), so these small molecules can exert their effects by inhibiting CDK8.

[0146] On this basis, cardiomyocytes (hiPSC-CMs) differentiated after the BI-1347 optimization system were identified. RNA-seq results showed that cardiomyocytes differentiated with or without BI-1347 treatment were similar, and cardiomyocyte-specific marker genes were significantly expressed (Figure 4a-c). Immunofluorescence results showed that the myocardium-specific expression of sarcomere-associated proteins cTNT, cTNI, and α-ACTININ was positive, and the myocardium-specific expression of transcription factors MEF2C and NKX2.5 was also positive, consistent with normal cardiomyocytes (Figure 4d). Electrophysiological identification results also showed that the cardiomyocytes differentiated with BI-1347 had relatively consistent characteristics such as action potential and calcium flow, and the proportions of each subtype of differentiated cardiomyocytes were similar (Figure 5a-j).

[0147] In summary, the addition of CDK8 inhibitors such as BI-1347 during the first stage of stem cell differentiation into cardiomyocytes (0-48 h) can expand the applicable range of CHIR concentration and time, and improve the efficiency and stability of the cardiomyocyte differentiation system between different cell lines or different batches.

Claims

1. A method for preparing cardiomyocytes by differentiating pluripotent stem cells, the method comprising: i) providing pluripotent stem cells; ii) culturing the pluripotent stem cells in a basal medium supplemented with a CDK8 inhibitor and a WNT signaling pathway activator for about 24 hours to about 48 hours, and then continuing to culture the cells in a basal medium without the CDK8 inhibitor and the WNT signaling pathway activator for about 24 hours; iii) culturing the cells obtained in step ii) in a basal medium supplemented with a WNT signaling pathway inhibitor for approximately 48 hours, and then continuing to culture the cells in a basal medium without the WNT signaling pathway inhibitor for approximately 24 hours; iv) culturing the cells obtained in step iii) in a basal medium supplemented with insulin for about 3-6 days; and v) Optionally, harvesting the obtained cardiomyocytes.

2. The method of claim 1, wherein the pluripotent stem cell is selected from embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC).

3. The method of claim 1 or 2, wherein the pluripotent stem cells in step i) are cultured in a multi-well plate.

4. The method of any one of claims 1 to 3, wherein the pluripotent stem cells in step i) are grown to a confluence of about 80-90%.

5. The method of any one of claims 1 to 4, wherein the basal culture medium is a combination of RPMI medium and insulin-free B27 cell culture supplement.

6. The method according to any one of claims 1 to 6, wherein the basal medium supplemented with insulin in step iv) is a combination of RPMI medium (such as RPMI1640 medium) and B27 cell culture supplement.

7. The method of any one of claims 1 to 6, wherein the CDK8 inhibitor is selected from BI-1347 or a structural analog thereof, MSC2530818 or a structural analog thereof, AS2863619 or a structural analog thereof, Senexin A or a structural analog thereof, CCT-251921 or a structural analog thereof, Senexin A or a structural analog thereof, C or a structural analog thereof, JH-XVI-178 or a structural analog thereof, CCT251545 or a structural analog thereof, BRD6989 or a structural analog thereof, JH-XI-10-02 or a structural analog thereof, SEL120-34A or a structural analog thereof, LY2857785 or a structural analog thereof, CDK8-IN-1 or a structural analog thereof, CDK8-IN-5 or a structural analog thereof, CDK8-IN-3 or a structural analog thereof, CDK8-IN-4 or a structural analog thereof, CDK8-IN-6 or a structural analog thereof, CDK8-IN-7 or a structural analog thereof, CDK8 / 19-IN-1 or a structural analog thereof, DS96432529 or a structural analog thereof, Wogonin or a structural analog thereof, preferably, the CDK8 inhibitor is BI-1347 or a structural analog thereof.

8. The method of any one of claims 1 to 7, wherein the WNT signaling pathway activator is CHIR99021 or a structural analog thereof, CHIR98014 or a structural analog thereof, or SB212763 or a structural analog thereof.

9. The method of any one of claims 1 to 8, wherein the WNT signaling pathway inhibitor is IWR1 or a structural analog thereof, IWP2 or a structural analog thereof, or IWP4 or a structural analog thereof.

10. The method of any one of claims 1-9, wherein the insulin is human insulin.

11. The method of any one of claims 1 to 10, wherein the final concentration of the CDK8 inhibitor, e.g., BI-1347, or a structural analog thereof, is about 0.001 μM to about 5 μM, preferably about 0.004 μM to about 3 μM, e.g., about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, or about 5 μM.

12. The method of any one of claims 1 to 11, wherein the final concentration of the WNT signaling pathway activator, such as CHIR99021 or a structural analog thereof, is about 1 μM to about 25 μM or higher, for example, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, greater than 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM or higher.

13. The method of any one of claims 1-12, wherein the concentration of the WNT signaling pathway inhibitor is about 1 μM to about 10 μM or higher, such as about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM or higher.

14. The method of any one of claims 1 to 13, wherein the pluripotent stem cells are human pluripotent stem cells, and the cardiomyocytes are human cardiomyocytes.

15. The method of any one of claims 1 to 14, wherein the cardiomyocytes are functional cardiomyocytes, such as cardiomyocytes capable of beating.

16. The method of any one of claims 1 to 15, wherein the cardiomyocytes express one or more cardiac-specific proteins selected from the group consisting of cTNT, cTNI, α-ACTININ, MEF2C, and NKX2.

5.

17. A cardiomyocyte prepared by the method according to any one of claims 1 to 16.

18. A pharmaceutical composition comprising cardiomyocytes prepared by the method according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

19. Use of the cardiomyocytes of claim 17 or the pharmaceutical composition of claim 18 in the preparation of a medicament for treating a cardiac disease in a subject in need thereof.

20. A method of treating cardiac disease in a subject in need thereof, comprising administering to the subject the cardiomyocytes of claim 17 or the pharmaceutical composition of claim 18.

21. The use of claim 19 or the method of claim 20, wherein the subject is a human.

22. Use of a CDK8 inhibitor in the preparation of a reagent for preparing cardiomyocytes by differentiating pluripotent stem cells.

23. The method of claim 22, wherein the CDK8 inhibitor is selected from BI-1347 or a structural analog thereof, MSC2530818 or a structural analog thereof, AS2863619 or a structural analog thereof, Senexin A or a structural analog thereof, CCT-251921 or a structural analog thereof, Senexin A or a structural analog thereof, C or a structural analog thereof, JH-XVI-178 or a structural analog thereof, CCT251545 or a structural analog thereof, BRD6989 or a structural analog thereof, JH-XI-10-02 or a structural analog thereof, SEL120-34A or a structural analog thereof, LY2857785 or a structural analog thereof, CDK8-IN-1 or a structural analog thereof, CDK8-IN-5 or a structural analog thereof, CDK8-IN-3 or a structural analog thereof, CDK8-IN-4 or a structural analog thereof, CDK8-IN-6 or a structural analog thereof, CDK8-IN-7 or a structural analog thereof, CDK8 / 19-IN-1 or a structural analog thereof, DS96432529 or a structural analog thereof, Wogonin or a structural analog thereof, preferably, the CDK8 inhibitor is BI-1347 or a structural analog thereof. 24 . The use according to claim 22 or 23 , wherein the agent is used in combination with a WNT signaling pathway activator, a WNT signaling pathway inhibitor and / or insulin to prepare cardiomyocytes by differentiation from pluripotent stem cells.

25. The use according to any one of claims 22 to 24, wherein the agent is used to improve the efficiency of preparing cardiomyocytes by differentiation from pluripotent stem cells. 26 . The use according to claim 22 , wherein the agent is used to improve the efficiency of preparing cardiomyocytes by differentiating pluripotent stem cells in the presence of a high concentration of a WNT signaling pathway activator.

27. The use according to any one of claims 22 to 26, wherein the reagent is used for preparing cardiomyocytes by the method according to any one of claims 1 to 16.

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